A method for modeling tree shrew hypertension

Abstract

The invention discloses a method for modeling a tree shrew hypertension model, comprising: (1) preparing a solvent: dissolving palmitic acid in anhydrous ethanol to prepare a 24 mg / ml storage solution for standby use; (2) diluting the storage solution in step (1) with 10% BSA to prepare a 6 mg / ml solution, and then administering the solution to the tree shrew by intraperitoneal injection at a dose of 20 mg / kg for 21 days to complete the modeling. The model preparation method is simple and has a high modeling rate.

Description

Technical Field

[0001] The invention belongs to the technical field of disease animal model construction, and in particular relates to a tree shrew hypertension model construction method. Background Art

[0002] Hypertension is a common cardiovascular disease. High blood pressure is also an important pathogenic factor for heart, kidney and cerebrovascular diseases. It can cause changes and damage to the structure and function of organs such as the heart, brain, kidneys, blood vessels, and fundus, significantly increasing the risk of related complications and seriously endangering human health.

[0003] Although in recent years, clinical blood pressure control for hypertensive patients has shown initial results, the threats to human health and social harm caused by its complications such as stroke and heart failure cannot be ignored. Hypertension has become the primary risk factor for increased incidence and mortality of cardiovascular disease among residents in my country. Therefore, hypertension has always been a key research topic in the cardiovascular field.

[0004] In order to better prevent and treat hypertension, explore the mechanism and efficacy of drugs for the treatment of hypertension, it is particularly important to understand and establish an animal model similar to the course of human hypertension. At present, animal models of hypertension mostly use rodents (rats and mice). Due to the large differences between them and humans, the research results obtained are often far from humans, resulting in the ultimate failure of drug development after a large amount of investment. Summary of the invention

[0005] The purpose of the present invention is to provide a method for modeling a tree shrew hypertension model.

[0006] The object of the present invention is achieved as follows: (1) Solvent preparation: palmitic acid is dissolved in anhydrous ethanol to prepare a 24 mg / ml storage solution for standby use; (2) the storage solution of step (1) is diluted with 10% BSA to a 6 mg / ml solution, and then the solution is intraperitoneally injected into tree shrews at a dose of 20 mg / kg for 21 days to complete the modeling.

[0007] The beneficial effects of the present invention are:

[0008] Tree shrews have the advantages of small size, easy reproduction, and fast reproduction. Some physiological characteristics of tree shrews, such as anatomical structure, nervous, immune and metabolic systems, are highly similar to those of primates including humans. Therefore, the present application successfully established a stable tree shrew hypertension model by intraperitoneal injection of palmitic acid at a dose of 20 mg / kg for 3 weeks. The model preparation method is simple and has a high model success rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The results of blood pressure changes of 5 groups of tree shrews during the experiment of this application;

[0010] Figure 2 Renin results of 5 groups of tree shrews during the experiment of this application;

[0011] Figure 3 The ACE results of 5 groups of tree shrews during the experiment of this application;

[0012] Figure 4 The NO results of 5 groups of tree shrews during the experiment of this application;

[0013] Figure 5 The NOS results of 5 groups of tree shrews during the experiment of this application;

[0014] Figure 6 These are the Ang Ⅱ results of 5 groups of tree shrews during the experiment of this application;

[0015] Figure 7 These are the ET-1 results of 5 groups of tree shrews during the experiment of this application. DETAILED DESCRIPTION

[0016] The technical solution of the present invention will be described clearly and completely in combination with the embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, but not all of them. All other embodiments belong to the protection scope of the present invention.

[0017] 1. Experimental Animals

[0018] Fifteen ordinary male Burmese tree shrews weighing 120-150 g were purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences [SCXK (Yunnan) K2023-0003] and kept in the ordinary animal room of Yunnan Luoyu Biotechnology Co., Ltd. [SYXK (Yunnan) K2021-0003] in stainless steel cages at room temperature (24 ± 2) °C, relative humidity (50 ± 5%, and light / dark cycle of 12 h / 12 ​​h).

[0019] 2. Main reagents

[0020] Reagent name company Palmitic acid Sigma BSA Solebao Normal saline Sichuan Kelun Pharmaceutical Anhydrous ethanol Xilong Chemical Renin Elisa Kit Wuhan Elerite Angiotensin Converting Enzyme Elisa Kit Wuhan Elerite Nitric Oxide Elisa Kit Wuhan Elerite Nitric Oxide Synthase Elisa Kit Wuhan Elerite Angiotensin II Elisa Kit Wuhan Elerite

[0021] 3. Main instruments

[0022] Instrument Name company Smart non-invasive blood pressure monitor Softron BP-98A Multifunctional ELISA reader Aosheng Feyond-A300

[0023] 4. Preparation of reagents

[0024] Palmitic acid solution: Palmitic acid is dissolved in anhydrous ethanol to prepare a 24 mg / ml storage solution. When used, it is diluted to 6 mg / ml with 10% BSA.

[0025] 5. Experimental methods

[0026] 5.1 Animal model preparation

[0027] Experimental groups: ①, normal group, ②, solvent group, ③, palmitic acid 5mg / kg group, ④, palmitic acid 10mg / kg group, ⑤, palmitic acid 20mg / kg group

[0028] Group ① received intraperitoneal injection of 500 μl of saline every day, Group ② received intraperitoneal injection of palmitic acid solvent (anhydrous ethanol + BSA solution) at the same time every day, and Groups ③-⑤ received intraperitoneal injection of palmitic acid according to the grouped dose. The injection time of the five groups lasted for 21 days. After the intraperitoneal injection, the rats continued to be fed.

[0029] 5.2 Arterial blood pressure measurement

[0030] After three days of adaptive feeding of tree shrews, blood pressure measurement adaptation experiments were performed on each tree shrew at least twice in the next four days to eliminate the changes in blood pressure caused by fear. After one week of adaptive feeding, the blood pressure of tree shrews was measured at 8:30-10:30 in the morning. After all tree shrews were measured, intraperitoneal injections were performed according to the groups, and the blood pressure of tree shrews was measured at the same time every week thereafter.

[0031] Blood pressure measurement method: Put the tree shrew head forward into the rat net, put it into the net bag, and expose the tail. After fixing the net bag, use electric clippers to shave the tail hair from 2 / 3 of the tail tip to the base of the tail, exposing the skin about 4 cm long at the base of the tail. Put the tree shrew's tail into the pressure sensor with a rubber membrane. After the tree shrew is quiet for 5 minutes, press the start button of the blood pressure meter to start automatic measurement. Each animal is measured again after an interval of 5 minutes, and a total of 3 measurements are taken, and the average value is taken as the final measurement value.

[0032] 5.3 Detection of Renin, Angiotensin Converting Enzyme (ACE), Nitric Oxide (NO), Nitric Oxide Synthase (NOS) and Angiotensin II (Ang II) Contents

[0033] Blood was collected from tree shrews before modeling (0d), 7d, 14d, 21d, 28d, and 35d, and plasma was separated after centrifugation. Elisa experiments were performed to detect the levels of renin (Renin), angiotensin converting enzyme (ACE), nitric oxide (NO), nitric oxide synthase (NOS), angiotensin II (AngⅡ), and endothelin-1 (ET-1).

[0034] 6. Experimental results

[0035] 6.1 Blood Pressure Results

[0036] The blood pressure (systolic and diastolic) of tree shrews in the normal group and the solvent group changed little on days 0, 7, 14, 21, 28, and 35. Compared with the normal group, the blood pressure (systolic and diastolic) of the palmitic acid group increased on days 7, 14, 21, 28, and 35 in a dose-dependent manner.

[0037] Table 1 Blood pressure changes of five groups of tree shrews during the experiment ( , n=3)

[0038] Grouping (n=3) 0d 7d 14d 21d 28d 35d Systolic blood pressure Normal group 124.00±8.54 124.33±6.81 120.33±4.73 125.33±8.39 122.67±6.51 123.00±9.17 Solvent Group 122.33±8.02 123.00±3.00 123.67±4.16 124.33±8.96 123.67±4.51 123.33±6.66 Palmitic acid 5mg / kg group 122.33±3.06 124.67±7.77 124.33±5.51 130.33±6.03 131.67±5.51 129.67±8.50 Palmitic acid 10mg / kg group 121.00±9.00 124.00±7.94 127.00±7.94 136.00±4.00 136.67±5.86 134.67±5.51 Palmitic acid 20 mg / kg group 123.33±8.50 129.33±4.16 140.33±5.13 146.33±11.93 145.33±13.50 146.67±9.29 Diastolic blood pressure Normal group 83.33±9.07 84.33±9.02 83.67±9.02 85.00±9.17 83.33±11.37 80.33±7.57 Solvent Group 84.00±9.85 83.00±8.19 83.33±11.93 81.00±9.85 83.67±5.51 82.00±9.54 Palmitic acid 5mg / kg group 84.00±8.00 83.00±9.64 81.00±4.36 89.67±6.11 90.67±7.51 88.00±4.58 Palmitic acid 10mg / kg group 82.67±8.74 84.33±9.07 89.67±5.69 94.33±9.50 92.67±7.51 96.67±10.79 Palmitic acid 20 mg / kg group 82.67±6.11 87.33±10.6 95.00±8.89 109.00±8.00 109.00±7.94 106.00±9.64

[0039] 6.2 Renin results

[0040] The plasma renin levels of tree shrews in the normal group and the solvent group remained basically the same on days 0, 7, 14, 21, 28, and 35, with little change. Compared with the normal group, the plasma renin levels in the palmitic acid group increased on days 7, 14, 21, 28, and 35 in a dose-dependent manner.

[0041] Table 2 Renin results of five groups of tree shrews during the experiment ( , n=3)

[0042] Grouping (n=3) 0d 7d 14d 21d 28d 35d Normal group 420.56±37.36 414.46±42.56 417.46±44.35 419.55±38.68 424.46±48.08 431.62±36.81 Solvent Group 413.59±28.19 424.57±39.47 417.57±34.78 424.55±42.02 425.31±59.15 422.40±52.94 Palmitic acid 5mg / kg group 423.36±55.04 416.53±38.21 424.46±48.08 440.59±40.83 442.30±63.02 439.56±43.58 Palmitic acid 10mg / kg group 418.53±38.67 428.46±49.86 444.58±40.57 503.86±41.13 496.93±48.00 506.82±37.20 Palmitic acid 20 mg / kg group 416.63±25.22 433.57±42.44 474.44±29.66 648.84±96.46 640.52±71.10 641.51±43.52

[0043] 6.3 ACE Results

[0044] The plasma ACE levels of tree shrews in the normal group and the solvent group remained basically the same on days 0, 7, 14, 21, 28, and 35, with little change. Compared with the normal group, the plasma ACE levels of the palmitic acid group increased on days 7, 14, 21, 28, and 35 in a dose-dependent manner.

[0045] Table 3 ACE results of five groups of tree shrews during the experiment ( , n=3)

[0046] Grouping (n=3) 0d 7d 14d 21d 28d 35d Normal group 3.42±0.23 3.33±0.21 3.52±0.18 3.4±0.18 3.37±0.26 3.4±0.22 Solvent Group 3.35±0.31 3.42±0.38 3.43±0.16 3.34±0.22 3.48±0.25 3.42±0.37 Palmitic acid 5mg / kg group 3.38±0.32 3.4±0.33 3.62±0.39 3.91±0.42 3.9±0.31 3.93±0.33 Palmitic acid 10mg / kg group 3.32±0.27 3.63±0.47 3.82±0.37 4.35±0.42 4.39±0.16 4.34±0.33 Palmitic acid 20 mg / kg group 3.33±0.28 3.78±0.42 4.74±0.46 7.62±1.00 7.85±0.68 7.67±1.04

[0047] 6.4 NO Result

[0048] The plasma nitric oxide levels of tree shrews in the normal group and the solvent group remained basically the same on days 0, 7, 14, 21, 28, and 35, with little change. Compared with the normal group, the plasma nitric oxide levels of the palmitic acid group showed a decreasing trend on days 7, 14, 21, 28, and 35, and were dose-dependent.

[0049] Table 4 NO results of five groups of tree shrews during the experiment ( , n=3)

[0050] Grouping (n=3) 0d 7d 14d 21d 28d 35d Normal group 3.36±0.18 3.36±0.36 3.26±0.36 3.36±0.36 3.36±0.48 3.26±0.48 Solvent Group 3.36±0.36 3.26±0.18 3.36±0.48 3.47±0.31 3.36±0.48 3.36±0.18 Palmitic acid 5mg / kg group 3.36±0.18 3.26±0.48 3.26±0.36 2.95±0.18 2.95±0.18 3.05±0.36 Palmitic acid 10mg / kg group 3.36±0.48 3.16±0.31 2.95±0.65 2.64±0.65 2.64±0.48 2.64±0.48 Palmitic acid 20 mg / kg group 3.47±0.31 2.84±0.31 2.43±0.48 2.22±0.31 2.11±0.48 2.22±0.31

[0051] 6.5 NOS Results

[0052] The plasma nitric oxide synthase levels of tree shrews in the normal group and the solvent group remained basically the same on days 0, 7, 14, 21, 28, and 35, with little change. Compared with the normal group, the plasma nitric oxide synthase levels in the palmitic acid group showed a decreasing trend on days 7, 14, 21, 28, and 35, and were dose-dependent.

[0053] Table 5 NOS results of five groups of tree shrews during the experiment ( , n=3)

[0054] Grouping (n=3) 0d 7d 14d 21d 28d 35d Normal group 872.52±51.16 859.22±65.46 876.36±25.96 857.29±73.89 866.87±43.36 857.44±31.28 Solvent Group 861.21±36.53 859.33±34.07 844.16±39.96 855.53±36.55 851.77±23.67 842.26±39.82 Palmitic acid 5mg / kg group 851.76±32.84 847.96±35.53 830.89±37.33 804.29±27.01 792.83±38.12 809.91±56.43 Palmitic acid 10mg / kg group 849.87±31.30 832.78±37.04 806.14±45.79 758.43±40.36 762.26±40.19 769.93±35.02 Palmitic acid 20 mg / kg group 847.96±37.29 815.59±62.69 785.18±45.38 679.44±50.04 689.07±58.14 692.27±60.83

[0055] 6.6 Ang II Results

[0056] The plasma angiotensin II levels of tree shrews in the normal group and the solvent group remained basically the same on days 0, 7, 14, 21, 28, and 35, with little change. Compared with the normal group, the plasma angiotensin II levels of the palmitic acid group increased on days 7, 14, 21, 28, and 35 in a dose-dependent manner.

[0057] Table 6 Ang Ⅱ results of five groups of tree shrews during the experiment ( , n=3)

[0058] Grouping (n=3) 0d 7d 14d 21d 28d 35d Normal group 536.63±39.60 538.41±34.93 547.65±49.74 543.75±38.04 553.48±33.05 546.52±17.96 Solvent Group 552.98±27.97 534.5±36.270 549.62±24.28 538.26±37.80 551.17±40.14 544.04±38.01 Palmitic acid 5mg / kg group 540.87±31.63 548.60±36.18 567.48±48.85 603.23±31.81 610.46±40.02 607.51±51.86 Palmitic acid 10mg / kg group 541.29±40.94 559.19±40.90 585.31±37.60 640.87±23.50 645.28±39.31 642.82±17.95 Palmitic acid 20 mg / kg group 544.79±28.09 574.52±44.41 638.83±34.74 740.06±42.30 740.19±58.90 744.40±41.61

[0059] 6.7 ET-1 Results

[0060] The plasma endothelin-1 levels of tree shrews in the normal group and the solvent group remained basically the same on days 0, 7, 14, 21, 28, and 35, with little change. Compared with the normal group, the plasma endothelin-1 levels of the palmitic acid group increased on days 7, 14, 21, 28, and 35 in a dose-dependent manner.

[0061] Table 7 ET-1 results of five groups of tree shrews during the experiment ( , n=3)

[0062] Grouping (n=3) 0d 7d 14d 21d 28d 35d Normal group 17.05±1.61 16.79±1.91 16.86±0.77 17.01±0.99 16.82±0.79 16.63±1.14 Solvent Group 16.82±0.89 16.75±1.25 17.01±0.69 16.9±1.65 16.75±1.1 17.08±0.56 Palmitic acid 5mg / kg group 16.70±0.45 16.74±0.64 18.77±1.56 22.14±2.62 21.90±2.48 22.30±2.62 Palmitic acid 10mg / kg group 17.05±1.33 17.92±1.10 20.31±1.23 24.89±1.34 24.25±1.88 25.06±1.80 Palmitic acid 20 mg / kg group 16.94±1.70 19.38±1.58 25.07±2.33 37.90±4.08 38.72±4.92 38.34±3.38

Claims

1. A method for modeling a tree shrew hypertension model, characterized in that: The steps include: (1) Solvent preparation: Dissolve palmitic acid in anhydrous ethanol to prepare a 24 mg / ml storage solution for later use; (2) The stock solution of step (1) was diluted with 10% BSA to a solution of 6 mg / ml, and then the solution was intraperitoneally injected into tree shrews at a dose of 20 mg / kg for 21 days to complete the modeling.

2. The method for establishing a tree shrew hypertension model according to claim 1, characterized in that: The tree shrew is a common male Burmese tree shrew.

3. The method for establishing a tree shrew hypertension model according to claim 1, characterized in that: The tree shrew has a body weight of 120-150 g, and is kept at a room temperature of 24±2° C., a relative humidity of 50±5%, and a light / dark cycle of 12 h / 12 ​​h.

Images